Bovine brucellosis is a disease associated with abortions and infertility, and is caused by the gram-negative organism Brucella abortus. Despite an active vaccination program, bovine brucellosis continues to be a problem in the United States (10, 16) and is an economically important disease. Numerous recent reports have been published on various vaccines for the prevention of experimental brucellosis in laboratory animals and cattle. These vaccines include live, reduced dose vaccines (1, 7, 25), nonviable whole cell vaccines (9, 11, 26, 27), cell-free detergent extracts (4, 5, 8, 13), phenol extracts (2, 18, 19), and ribosomal preparations (17). Some of the preparations, in particular, the detergent-extractable protein-peptidoglycan fraction, provide durable immunity in mioe and young cattle, but were less efficacious in adult cattle (6). Inclusion of adjuvants such a trehalose dimycolate (TDM) and muramyl dipeptide (MDP) has been shown to enhance the efficacy of some bacterins (26). The use of complex heterogeneous o killed-cell vaccines often results in the production of undesirable granulomas and undesirable long-lasting serum antibody titers which interfere with the diagnostic tests used for bovine brucellosis. This latter problem is also occasionally encountered with live vaccines. Nonviable vaccines are desirable, since the live attenuated vaccine strain can cause mammary gland infections and also abortions in adult vaccinated cattle. However, many of the heterogeneous multicomponent vaccine preparations such as lipopolysaccharides (LPS) and proteins may result in antagonistic immune responses to the vaccine (6). To avoid these problems, it is preferable to develop vaccine preparations which are biochemically homogeneous. One approach to this goal has led to the development of a method (20) for the isolation of a fully soluble and stable protein preparation (BCS protein) from Brucella abortus strain 19.
It has been previously shown that a mixture of proteins can be removed from the surface of methanol-inactivated Brucella abortus cells by aqueous hypertonic sodium chloride-sodium citrate (20). Such a protein mixture isolated from an attenuated Brucella abortus strain was immunogenic in both rodents and appeared to be immunogenic in cattle. One of the proteins, having a molecular weight of around 30,000, was purified by chromatofocusing on a PBE column developed with a descending pH gradient from pH 6.2 to 4.0. The purified protein was found to be a more effective immunogen in lemmings than the total multicomponent extract (25). However, the isolation procedure employed (i.e., chromatofocusing) did not permit recovery of the protein in sufficient quantity for use in preparing an antiserum.
As described in Tabatabai, et al. (1979, Ref. 20) the washed methanol-inactivated cells were resuspended in 1 M NaCl-0.1 M sodium citrate and agitated with glass beads in a tissue disintegrator. The protein in the supernatant thus obtained was precipitated with ammonium sulfate to obtain the protein mixture. Polyacrylamide gel electrophoresis indicated the presence of a protein of approximate molecular weight of 30,000 daltons (Ref. 20, FIG. 4, p. 674), but the procedure was not practical for use in preparing antiserum to the protein.
It has been suggested that soluble salt-extractable proteins from Brucella abortus may be of potential value for preparing vaccines and/or for use as diagnostic agents in the prevention or diagnosis of bovine brucellosis (Tabatabai, et al., 1984, Ref. 22). Further, the salt-extractable proteins of Brucella abortus have been analyzed by crossed immuno-electrophoresis using rabbit antiserum protein antigens and by isoelectric focusing with polyacrylamide gels Tabatabai, et al. 1984, Ref. 23). The isoelectric pH's of the extracted proteins were profiled in FIG. 4, page 557, of Ref. 23.